- Email: [email protected]
The Correlation of Prostate Volume and Prostate-specific Antigen Levels With Positive Bacterial Prostate Tissue Cultures
Accepted Manuscript Title: The Correlation of Prostate Volume and PSA Levels with Positive Bacterial Prostate Tissue Cultures Author: Stefan Heidler, Martin Drerup, Lukas Lusuardi, Ursula Bannert, Katharina Bretterbauer, Johannes Bures, Franz Dietersdorfer, Eva DlouhySchütz, Clemens Hessler, Rainer Karpf, Lie-Anna Mittellehner, Barbara Mitlöhner, Stephan Schwarz, Günther Thomay, Georg Lösch, Christa Freibauer, Walter Albrecht PII: DOI: Reference:
S0090-4295(18)30194-8 https://doi.org/10.1016/j.urology.2018.02.035 URL 20931
To appear in:
Urology
Received date: Accepted date:
10-11-2017 26-2-2018
Please cite this article as: Stefan Heidler, Martin Drerup, Lukas Lusuardi, Ursula Bannert, Katharina Bretterbauer, Johannes Bures, Franz Dietersdorfer, Eva Dlouhy-Schütz, Clemens Hessler, Rainer Karpf, Lie-Anna Mittellehner, Barbara Mitlöhner, Stephan Schwarz, Günther Thomay, Georg Lösch, Christa Freibauer, Walter Albrecht, The Correlation of Prostate Volume and PSA Levels with Positive Bacterial Prostate Tissue Cultures, Urology (2018), https://doi.org/10.1016/j.urology.2018.02.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The correlation of prostate volume and PSA levels with positive bacterial prostate tissue cultures Stefan Heidlera, Martin Drerupb, Lukas Lusuardib, Ursula Bannert a, Katharina Bretterbauer a, Johannes Bures a, Franz Dietersdorfer a, Eva Dlouhy-Schütz a, Clemens Hessler a, Rainer Karpf a, Lie-Anna Mittellehner a, Barbara Mitlöhner a, Stephan Schwarza, Günther Thomay a, Georg Lösch c, Christa Freibauer c, and Walter Albrechta
a
Department of Urology, Krankenhaus Mistelbach, Austria
b
Department of Urology, Paracelsus Medical University Salzburg, Austria c
Department of Pathology, Krankenhaus Mistelbach, Austria
Corresponding author: Stefan Heidler, MD, F.E.B.U. Department of Urology Krankenhaus Mistelbach Liechtensteinstrasse 67 2130 Mistelbach Austria Phone: +43 2572 9004 21904 FAX: +43 2572 9004 49251 E-Mail: [email protected]
Keywords: bacteria, prostate gland tissue culture, prostate volume, BPE, BPH, PSA
Word count of the text: 2446 Word count of the abstract: 142
1
Page 1 of 20
Abstract Objective: To compare prostate volume and PSA levels with bacterial growth in prostate tissue cultures. Methods: 50 male patients who underwent transuretheral prostate resection (TURP) were investigated prospectively. Resection chips from the prostate gland were added to Brain Heart Infusion Medium and incubated. PSA levels were determinded preoperatively at our urology ward. The prostate gland volume was estimated by transabdominal ultrasound (TAUS) examination preoperatively. Results: Persons with positive bacterial prostate tissue cultures have a greater prostate volume. This is significant in patients with and without histopathological signs of prostatitis. Persons with positive bacterial prostate tissue cultures have higher PSA values. This is significant in patients without histopathological signs of prostatitis. Conclusions: People with positive bacterial prostatic tissue culture have a higher prostate volume in comparison to patients with negative culture findings and show a tendency towards increased PSA levels as well.
Introduction: There is widespread consensus in literature, that the size of the prostate gland increases throughout a man’s lifetime [1,2]. Benign prostatic enlargement (BPE) and benign prostatic hyperplasia (BPH) is a common disorder affecting 50-80% of the aged male population. Factors leading to prostate gland growth have been the focus of many studies already. The correlation between the size of the prostate gland and patient age is sufficiently proven in many publications. Therefore, BPE is considered a progressive disease and its greatest risk factor for progression is age [3]. A big 2
Page 2 of 20
number of studies have revealed a characteristic pattern of human prostatic growth with age [4,5,6]. It has already been reported that the average percentage of total prostate volume (TPV) and transition zone volume (TZV) increases per year of follow-up at 2.2% and 3.5%, respectively [4]. A strong correlation exists between serum prostate-specific antigen (PSA) levels and prostate volume as well [7]. Androgens and age have been traditionally considered the main determinants of prostate enlargement, but in the last years a potentially important role of chronic inflammation in BPH pathogenesis has emerged [8]. In the peer review literature, a large number of studies already prove that a chronic prostate inflammation can be linked to the development and progress not only of BPH, but also of prostate cancer [9,10,11,12,13]. In our most recent study we were able to demonstrate a wide variety of bacteria in prostate gland tissue, which was associated with an increased complication rate in patients undergoing TUR [14]. The hypothesis of the influence of inflammatory processes on prostate gland enlargement ought to be easily reproductable. Tissue samples acquired during TURP are suited perfectly for histological and bacterial analysis, as they enable proof of bacterial growth in a timely and cost effective manner. The aim of this study was to compare prostate volume and PSA levels with bacterial growth in prostate tissue cultures for the very first time.
3
Page 3 of 20
Material and Methods: 50 male patients undergoing transuretheral prostate resection (TURP) were investigated prospectively. This study was approved by the Ethics Committee of the Medical University of Salzburg. PSA levels were determinded preoperatively at our urology ward. The prostate gland volume was estimated by transabdominal ultrasound (TAUS) examination at our department with a 6-MHz curved array transducer (BK medical profocus 2202), which was performed by an urologist. Prostate width (maximal transverse diameter) was measured on an axial image, while prostate length (the longitudinal diameter as defined by the distance between proximal external sphincter and urinary bladder) and height (maximal anteroposterior diameter) were measured on a mid-sagittal image. The total prostate volume was calculated with the prolate elliptical formula, π/6×width×height×length [15]. We used Columbia Agarplates and McConkey Agarplates for culturing. Intrasurgically, an average of two prostate tissue resection chips from a deeper layer of the prostate gland were taken under aseptic conditions, were added to Brain Heart Infusion Medium and then incubated at temperatures of 34 to 36 degrees Celsius. Results were visible from 48 hours on. Prostate resection chips were then sent to the pathological department for histopathological examination. There, the resection chips were embedded in paraffin, an average of 10 blocks per patient, corresponding with an average resection weight of 30 grams. If a carcinoma was found in one or more of these 10 blocks, all remaining chips were embedded and examined. Prostatitis was diagnosed, when leukocytes in the interstitium and in the glandular cells were detected by the pathologist. Chronic inflammatory processes were defined by plasmatic cells, lymphocytes and an atrophy of the glandular cells.
4
Page 4 of 20
We included patients that were anamnestically infection free for the least 4 weeks prior to surgery. Patients with indwelling transurethral catheters were excluded in the study. We evaluated the correlation between a positive bacterial culture of the prostate gland tissue and a larger prostate volume as well as a positive bacterial culture of the prostate gland and PSA levels based on following hypotheses: Hypothesis 1: positive bacterial cultures of the prostate are associated with a higher PSA and larger prostate volume in patients, with or without histopathological signs of prostatitis excluding patients with prostate cancer. Hypothesis 2a: positive bacterial cultures of the prostate are associated with higher PSA levels and larger prostate volumes in patients without histopathological signs of prostatitis. Hypothesis 2b: positive bacterial cultures of the prostate are associated with higher PSA levels and larger prostate volume in patients with histopathological signs of prostatitis. Hypothesis 2c: bacterial cultures of the prostate are associated with higher PSA levels and a larger prostate volume in patients with prostate cancer. Hypothesis 3: positive bacterial cultures are associated with larger prostate volume in patients with or without histopathological signs of prostatitis or prostate cancer.
Statistical Analysis: The analysis of the data was carried out using the program IBM SPSS Statistics 19.0. According to current scientific practice, the significance level is set to α = 0.05, that is, there is a 5% probability of error in hypothesis testing. A hypothesis thus always holds as assumed if the reported pvalue is <.05. The Point-biserial correlation coefficient test (Pearson correlation coefficient) has been used. 5
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Results: The sample comprises a total of n = 50 persons. The mean age of the subjects examined was M = 70,92 years (SD = 9,10). Selected sample values are illustrated in Table 1 [Table 1]. Of the total sample, 46% (n = 23) had a BPH with prostatitis, 40% (n = 20) had BPH without prostatitis and 14% (n = 7) had a prostate carcinoma. While 34% (n = 17) of the 50 people had a positive bacterial culture [Table 2], a negative culture result was found in 66% of the samples (n =33). Of the patients who had a BPH with prostatitis (n =23), 10 Patients had a positive bacterial culture (43,5%). Of the patients who had a BPH without prostatitis (n =20), 4 Patients had a positive bacterial culture (20%). Of the patients diagnosed with prostate cancer (n =7), 3 Patients had a positive bacterial culture (42,9%). Hypothesis 1: positive bacterial cultures of the prostate gland are associated with a higher PSA and a larger prostate volume in patients, with or without histopathological signs of prostatitis, excluding patients with prostate cancer. A significant result was obtained for PSA values (r = 0,35, p = 0,02) as well as for the prostate volume (r = 0,43, p = 0,002). Persons with positive bacterial cultures have higher PSA values and have a greater prostate volume. This is significant in patients with and without histopathological signs of prostatitis. Hypothesis 1 can thus be confirmed [Figure 1, Figure 2, Table 1].
Hypothesis 2a: positive bacterial cultures of the prostate are associated with a higher PSA and a larger prostate volume in patients without histopathological signs of prostatitis. There were significant results for the PSA values (r = 0,70, p <0,001) as well as for the prostate volume (r = 0,49, p = 0,01). People with positive bacterial findings have higher PSA values and larger prostate volume in patients without histopathological signs of prostatitis. The results confirm the significant correlation, so hypothesis 2a can be accepted. 6
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Hypothesis 2b: positive bacterial cultures of the prostate are associated with higher PSA levels and larger prostate volumes in patients with histopathological signs of prostatitis. While the PSA values did not show a significant correlation with the prostate tissue culture (r = 0,15, p = 0,26) in patients with histopathological signs of prostatitis, a significant correlation was found with the prostate volume (r = 0,51, p = 0,01) in patients with histopathological signs of prostatitis. Correspondingly, hypothesis 2b can only be accepted with restrictions. Hypothesis 2c: bacterial cultures of the prostate are associated with higher PSA levels and a larger prostate volume in patients with prostate cancer. This did not produce any significant results for PSA levels (r = 0,4, p = 0,47), nor for prostate volume (r = 0,0, p = 0,50) in patients with prostate cancer. The results indicate that hypothesis 2c cannot be assumed to be valid. However, since in this case only few patient data were available, we realize that this assumption is statistically underpowered. It is quite possible that an effect exists, but the existing data are not sufficient to validate this. Hypothesis 3: positive bacterial cultures are associated with larger prostate volume in patients with or without histopathological signs of prostatitis, including patients with prostate cancer. There was a significant result for the prostate volume (r = 0,38, p = 0,004). People with positive bacterial findings have a higher prostate volume in comparison to the healthy male population. The correlation is significant, hypothesis 3 is thus to be confirmed.
Comment:
7
Page 7 of 20
First of all, a note on the ultrasound examination. We realize that some authors prefer transrectal ultrasound (TRUS) for prostate volume measurement. We opted for suprapubic, transabdominal ultrasound (TAUS) however for several reasons. Even though
transrectal
ultrasound
has
shown
to
be
superior
to
suprapubic
(transabdominal) volume measurement [16,17], TAUS is a widely accepted method of imaging the prostate worldwide. This is because it is more convenient for the patient and the urologist. TRUS on the other hand is associated with some level of resistance due to its invasiveness and associated discomfort [18]. Huang confirmed in 2004 that no statistically significant differences were found between the transabdominal ultrasound as compared to transrectal sonography [19].
The strength of our study are the easily reproducible results obtained by simple and safe examination methods. Our study shows that the bacterial colonization correlates strongly with a higher prostate gland volume in general. There is also a correlation concerning PSA levels, but in patients with histopathological signs of prostatitis without a positive tissue culture, PSA is elevated, too. As already mentioned in the introduction, it is well known the PSA correlates with prostate volume [7]. The correlation between prostate volume and positive tissue cultures has not been described in this form yet, but does not necessarily prove causation. Although this might be the case, the correlations noted in our study could also reflect a competing hypothesis that BPH growth leads to higher levels of PSA and that BPH growth makes the prostate a larger target for infection. At this stage, the uncertainty remains whether bacterial colonization is a consequence of enlargement, or whether it is the main cause. Further investigation into causation will require additional studies.
8
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Patients with chronic inflammation and benign prostatic hyperplasia (BPH) have been shown to have larger prostate volumes, with more severe lower urinary tract symptoms (LUTS) and a higher probability of acute urinary retention than their counterparts without inflammation [20]. Although the pathogenesis of BPH is not yet fully understood and several mechanisms seem to be involved in the development and progression, recent studies strongly suggest that BPH is an immune inflammatory disease. The T-cell activity and associated autoimmune reaction seem to induce epithelial and stromal cell proliferation [21]. In this context, the hypothesis published by Roper about the development of BPH opens an interesting field for discussion: the enzyme Phospholipase D (PLD) which is attached to the outer membrane of Escherichia coli (E. coli) is the basic cause of BPH. It has been asserted that repeated colonization by E. coli of the transitional zone of the prostate gland and the release of PLD following repeated destruction of these colonized bacteria leads to prostate enlargement. Also involved are a variety of interleukins and other inflammatory cell cytokines, secreted by the stroma, which may further promote autocrine/paracrine proliferation of BPH cells. [22]. Several different stimuli may induce chronic prostatic inflammation, which in turn could lead to tissue damage and continuous wound healing, thus contributing to prostatic enlargement. In our study cohort, lack of proof of bacterial colonization does not necessarily prove the absence of bacterial colonization as such. Some bacteria probably can´t be detected conventionally. Interestingly, in 2012, a study group reported that they had detected bacteria in women's urine, which remained unobserved in standard tests. The standard urine culture does not detect slow-growing bacteria that will not survive in the presence of oxygen. In contrast to previously known germs, which are routinely found in urine samples, for example, in the case of urinary bladder infections, these 9
Page 9 of 20
bacteria would not grow in the laboratory under normal conditions, which explains why they have been overlooked in urine analyzes. They detected the microorganisms by genetic analysis [23]. In addition to our findings, an enlarged prostate can also be seen in patients with positive prostate tissue culture, even in the absence of inflammatory signs of the tissue. Thus, the inflammatory stimulus predescribed in the cited studies above cannot be exclusively responsible for an enlargement of the prostate gland. Other pathways of liberation of growth factors due to bacterial colonization might also be responsible. So far, no study data exists whether certain types of bacteria might have a positive, negative or no effect on the prostate gland and the direct connection between bacterial colonization and clinical symptoms is yet to be made. Recently, a study group from Milano, Italy, states the importance of the prostatic microbiome of the prostate tumor microenvironment, which contains a plethora of bacteria. The bacteria set themselves within the gland with a distribution dependent on the nature of the tissue, thus suggesting a possible pathophysiological correlation between the composition of the local microbial niche and the presence of the tumor itself. The role of specific bacteria and their potential to be exploited as new biomarkers remains to be investigated further [24] and could be a predictor of response to treatment as well. Thus, the ability to identify patients with a high risk for BPH progression and cancer genesis may be crucial to prevent aggravation of symptoms, genesis of prostate cancer and eventually develop target therapies. The ability of the immune system to fight diseases could depend on the urogenital microbiome. Although research on the urogenital tract are still scarce, initial studies have shown that the microbial populations in urine, seminal fluid, and prostatic fluid are significantly different in men with BPH from men with prostate cancer. An important goal for the future is to learn more about its role in disease development by 10
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researching the microbiome of the genitourinary tract. Some particular colonization types might even offer protection against recurrent infections, prostate enlargement, LUTS or even cancer. Keeping in mind the rapidly growing antibiotic resistance worldwide, it is important to identify true uropathogens to be able to optimize antibacterial therapy.
11
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Conclusions: People with positive bacterial prostatic tissue culture have a higher prostate volume in comparison to patients with negative culture findings. Persons with positive bacterial prostate tissue cultures show a tendency towards higher PSA levels.
12
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References: [1] Berry SJ, Coffey DS, Walsh PC, et al. The development of human benign prostatic hyperplasia with age. J Urol 1984;132:474–9. [2] Xia SJ, Xu XX, Teng JB, et al. Characteristic pattern of human prostatic growth with age. Asian J Androl 2002;4:269–71. [3] Emberton M, Andriole GL, de la Rosette J, et al. Benign prostatic hyperplasia: a progressive disease of aging men. Urology 2003;61:267–73. [4] Bosch JL, Tilling K, Bohnen AM, et al. Establishing normal reference ranges for prostate volume change with age in the population-based Krimpen-study: prediction of future prostate volume in individual men. Prostate 2007;67:1816–24. [5] Rhodes T, Girman CJ, Jacobsen SJ, et al. Longitudinal prostate growth rates during 5 years in randomly selected community men 40 to 79 years old. J Urol 1999;161:1174–9. [6] Loeb S, Kettermann A, Carter HB, et al. Prostate volume changes over time: results from the Baltimore Longitudinal Study of Aging. J Urol 2009;182:1458–62. [7] Roehrborn CG, Boyle P, Gould AL, et al. Serum prostate-specific antigen as a predictor of prostate volume in men with benign prostatic hyperplasia. Urology 1999;53:581-9. [8] Fibbi B, Penna G, Morelli A, et al. Chronic inflammation in the pathogenesis of benign prostatic hyperplasia. Int J Androl 2010;33:475-88.
13
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[9] De Nunzio C, Kramer G, Marberger M, et al. The controversial relationship between benign prostatic hyperplasia and prostate cancer: the role of inflammation. Eur Urol 2011;60:106-17. [10] De Marzo AM, Platz EA, Sutcliffe S, et al. Inflammation in prostate carcinogenesis. Nat Rev Cancer 2007;7:256–269. [11] Vasto S, Carruba G, Candore G, et al. Inflammation and prostate cancer. Future Oncol 2008;4:637–645. [12] Cohen RJ, Shannon BA, McNeal JE, et al. Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: a possible link to cancer evolution? J Urol 2005;173:1969–1974. [13] Alexeyev OA, Marklund I, Shannon B, et al. Direct visualization of Propionibacterium acnes in prostate tissue by multicolor fluorescent in situ hybridization assay. J Clin Microbiol 2007;45:3721–3728. [14] Heidler S, Bretterbauer K, Schwarz S, et al. Diversity of Bacterial Urine and Prostate Gland Tissue Cultures in Patients Undergoing Transurethral Prostate Gland Resection. Urol Int 2016;97:336-339. [15] Aarnink RG, de la Rosette JJ, Debruyne FM, et al. Formula-derived prostate volume determination. Eur Urol 1996;29:399–402. [16] Loch AC, Bannowsky A, Baeurle L, et al. Technical and anatomical essentials for transrectal ultrasound of the prostate. World J Urol 2007;25: 361.
14
Page 14 of 20
[17] Stravodimos KG, Petrolekas A, Kapetanakis T, et al. TRUS versus transabdominal ultrasound as a predictor of enucleated adenoma weight in patients with BPH: a tool for standard preoperative work-up? Int Urol Nephrol 2009;41:767. [18] Park SB, Kim JK, Choi SH, et al. Prostate volume measurement by TRUS using heights obtained by transaxial and midsagittal scanning: comparison with specimen volume following radical prostatectomy. Korean J Radiol 2000;1:110-3. [19] Huang Foen Chung JW, de Vries SH, Raaijmakers R, Postma R et al. Prostate volume ultrasonography: the influence of transabdominal versus transrectal approach, device type and operator. Eur Urol 2004;46:352-6. [20] Gandaglia G, Briganti A, Gontero P, et al. The role of chronic prostatic inflammation in the pathogenesis and progression of benign prostatic hyperplasia (BPH). BJU Int 2013;112:432-41. [21] Bostanci Y, Kazzazi A, Momtahen S, Laze J, et al. Correlation between benign prostatic hyperplasia and inflammation.Curr Opin Urol 2013;23:5-10. [22] Roper WG. The prevention of benign prostatic hyperplasia (bph).Med Hypotheses 2017 Mar;100:4-9. [23] Wolfe AJ, Toh E, Shibata N, et al. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol 2012;50:1376-83. [24] Cavarretta I, Ferrarese R, Cazzaniga W, et al. The Microbiome of the Prostate Tumor Microenvironment. Eur Urol 2017;72:625-631.
15
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Figure 1: PSA and positive prostate gland tissue culture Figure 2: prostate volume and positive prostate gland tissue culture
16
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Table 1: study group data All Patients (n=50)
Prostatitis (n=30)
No prostatitis (n=20)
Age
Vol.
PSA
Age
Vol.
PSA
Age
Vol.
PSA
Positive tissue culture (n=17) Age Vol. PSA
Negative tissue culture (n=33) Age Vol. PSA
Mean (M)
70,9
49,9
4,2
72.2
43.4*
3.9
69.3
55.6*
2.88*
72.7
62.3*
4.9
70.2
42.9
2.9
Median (Mdn) Stan-darddevi-ation (SD) Mini-mum (Min) Maxi-mum (Max) Vol.: *p<.05:
71,5
50
3,2
73.0
40.0
3.4
68.0
55.0
2.15
74
50
4.3
70
40
2.2
9,1
22,6
3,9
8.6
22.5
3.2
8.2
23.7
2.22
8.8
28.4
3.1
8.4
19.1
2.6
48
15
0,2
59.0
15.0
0.4
60.0
20.0
0.50
59
15
0.6
59
20
0.4
88
100
17,7
88.0
100
12
86
100
9.30
87
100
9.9
88
80
12
prostate volume statistical significance
17
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Table 2: Types of bacteria in prostate gland tissue Patients Bacteria 1
Staphylococcus saprophyticus
2
Escherichia coli
3
Staphylococcus aureus
4
Pseudomonas aeruginosa
5
Enterococcus faecalis
6
Staphylococcus epidermidis
7
Corynebakterium Glucuronolyticum
8
Escherichia coli (AmpC) - MRE
9
Staphylococcus aureus
10
Enterobacter cloace
11
Staphylococcus aureus
12
Corynebacterium glucuronolyticum
13
Enterococcus faecalis
14
Staphylococcus epidermidis and Koagulase negativ
15
Enterococcus faecalis
16
Staphylococcus epidermidis
17
Escherichia coli
18
Page 18 of 20
Figure 1
20
15
PSA
10
5
0 positiv e
negativ prostatic tissue culture
19
Page 19 of 20
Figure 2
100
80
60 Prostate volume 40
20
0 positiv e
negativ prostatic tissue culture
20
Page 20 of 20
S0090-4295(18)30194-8 https://doi.org/10.1016/j.urology.2018.02.035 URL 20931
To appear in:
Urology
Received date: Accepted date:
10-11-2017 26-2-2018
Please cite this article as: Stefan Heidler, Martin Drerup, Lukas Lusuardi, Ursula Bannert, Katharina Bretterbauer, Johannes Bures, Franz Dietersdorfer, Eva Dlouhy-Schütz, Clemens Hessler, Rainer Karpf, Lie-Anna Mittellehner, Barbara Mitlöhner, Stephan Schwarz, Günther Thomay, Georg Lösch, Christa Freibauer, Walter Albrecht, The Correlation of Prostate Volume and PSA Levels with Positive Bacterial Prostate Tissue Cultures, Urology (2018), https://doi.org/10.1016/j.urology.2018.02.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The correlation of prostate volume and PSA levels with positive bacterial prostate tissue cultures Stefan Heidlera, Martin Drerupb, Lukas Lusuardib, Ursula Bannert a, Katharina Bretterbauer a, Johannes Bures a, Franz Dietersdorfer a, Eva Dlouhy-Schütz a, Clemens Hessler a, Rainer Karpf a, Lie-Anna Mittellehner a, Barbara Mitlöhner a, Stephan Schwarza, Günther Thomay a, Georg Lösch c, Christa Freibauer c, and Walter Albrechta
a
Department of Urology, Krankenhaus Mistelbach, Austria
b
Department of Urology, Paracelsus Medical University Salzburg, Austria c
Department of Pathology, Krankenhaus Mistelbach, Austria
Corresponding author: Stefan Heidler, MD, F.E.B.U. Department of Urology Krankenhaus Mistelbach Liechtensteinstrasse 67 2130 Mistelbach Austria Phone: +43 2572 9004 21904 FAX: +43 2572 9004 49251 E-Mail: [email protected]
Keywords: bacteria, prostate gland tissue culture, prostate volume, BPE, BPH, PSA
Word count of the text: 2446 Word count of the abstract: 142
1
Page 1 of 20
Abstract Objective: To compare prostate volume and PSA levels with bacterial growth in prostate tissue cultures. Methods: 50 male patients who underwent transuretheral prostate resection (TURP) were investigated prospectively. Resection chips from the prostate gland were added to Brain Heart Infusion Medium and incubated. PSA levels were determinded preoperatively at our urology ward. The prostate gland volume was estimated by transabdominal ultrasound (TAUS) examination preoperatively. Results: Persons with positive bacterial prostate tissue cultures have a greater prostate volume. This is significant in patients with and without histopathological signs of prostatitis. Persons with positive bacterial prostate tissue cultures have higher PSA values. This is significant in patients without histopathological signs of prostatitis. Conclusions: People with positive bacterial prostatic tissue culture have a higher prostate volume in comparison to patients with negative culture findings and show a tendency towards increased PSA levels as well.
Introduction: There is widespread consensus in literature, that the size of the prostate gland increases throughout a man’s lifetime [1,2]. Benign prostatic enlargement (BPE) and benign prostatic hyperplasia (BPH) is a common disorder affecting 50-80% of the aged male population. Factors leading to prostate gland growth have been the focus of many studies already. The correlation between the size of the prostate gland and patient age is sufficiently proven in many publications. Therefore, BPE is considered a progressive disease and its greatest risk factor for progression is age [3]. A big 2
Page 2 of 20
number of studies have revealed a characteristic pattern of human prostatic growth with age [4,5,6]. It has already been reported that the average percentage of total prostate volume (TPV) and transition zone volume (TZV) increases per year of follow-up at 2.2% and 3.5%, respectively [4]. A strong correlation exists between serum prostate-specific antigen (PSA) levels and prostate volume as well [7]. Androgens and age have been traditionally considered the main determinants of prostate enlargement, but in the last years a potentially important role of chronic inflammation in BPH pathogenesis has emerged [8]. In the peer review literature, a large number of studies already prove that a chronic prostate inflammation can be linked to the development and progress not only of BPH, but also of prostate cancer [9,10,11,12,13]. In our most recent study we were able to demonstrate a wide variety of bacteria in prostate gland tissue, which was associated with an increased complication rate in patients undergoing TUR [14]. The hypothesis of the influence of inflammatory processes on prostate gland enlargement ought to be easily reproductable. Tissue samples acquired during TURP are suited perfectly for histological and bacterial analysis, as they enable proof of bacterial growth in a timely and cost effective manner. The aim of this study was to compare prostate volume and PSA levels with bacterial growth in prostate tissue cultures for the very first time.
3
Page 3 of 20
Material and Methods: 50 male patients undergoing transuretheral prostate resection (TURP) were investigated prospectively. This study was approved by the Ethics Committee of the Medical University of Salzburg. PSA levels were determinded preoperatively at our urology ward. The prostate gland volume was estimated by transabdominal ultrasound (TAUS) examination at our department with a 6-MHz curved array transducer (BK medical profocus 2202), which was performed by an urologist. Prostate width (maximal transverse diameter) was measured on an axial image, while prostate length (the longitudinal diameter as defined by the distance between proximal external sphincter and urinary bladder) and height (maximal anteroposterior diameter) were measured on a mid-sagittal image. The total prostate volume was calculated with the prolate elliptical formula, π/6×width×height×length [15]. We used Columbia Agarplates and McConkey Agarplates for culturing. Intrasurgically, an average of two prostate tissue resection chips from a deeper layer of the prostate gland were taken under aseptic conditions, were added to Brain Heart Infusion Medium and then incubated at temperatures of 34 to 36 degrees Celsius. Results were visible from 48 hours on. Prostate resection chips were then sent to the pathological department for histopathological examination. There, the resection chips were embedded in paraffin, an average of 10 blocks per patient, corresponding with an average resection weight of 30 grams. If a carcinoma was found in one or more of these 10 blocks, all remaining chips were embedded and examined. Prostatitis was diagnosed, when leukocytes in the interstitium and in the glandular cells were detected by the pathologist. Chronic inflammatory processes were defined by plasmatic cells, lymphocytes and an atrophy of the glandular cells.
4
Page 4 of 20
We included patients that were anamnestically infection free for the least 4 weeks prior to surgery. Patients with indwelling transurethral catheters were excluded in the study. We evaluated the correlation between a positive bacterial culture of the prostate gland tissue and a larger prostate volume as well as a positive bacterial culture of the prostate gland and PSA levels based on following hypotheses: Hypothesis 1: positive bacterial cultures of the prostate are associated with a higher PSA and larger prostate volume in patients, with or without histopathological signs of prostatitis excluding patients with prostate cancer. Hypothesis 2a: positive bacterial cultures of the prostate are associated with higher PSA levels and larger prostate volumes in patients without histopathological signs of prostatitis. Hypothesis 2b: positive bacterial cultures of the prostate are associated with higher PSA levels and larger prostate volume in patients with histopathological signs of prostatitis. Hypothesis 2c: bacterial cultures of the prostate are associated with higher PSA levels and a larger prostate volume in patients with prostate cancer. Hypothesis 3: positive bacterial cultures are associated with larger prostate volume in patients with or without histopathological signs of prostatitis or prostate cancer.
Statistical Analysis: The analysis of the data was carried out using the program IBM SPSS Statistics 19.0. According to current scientific practice, the significance level is set to α = 0.05, that is, there is a 5% probability of error in hypothesis testing. A hypothesis thus always holds as assumed if the reported pvalue is <.05. The Point-biserial correlation coefficient test (Pearson correlation coefficient) has been used. 5
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Results: The sample comprises a total of n = 50 persons. The mean age of the subjects examined was M = 70,92 years (SD = 9,10). Selected sample values are illustrated in Table 1 [Table 1]. Of the total sample, 46% (n = 23) had a BPH with prostatitis, 40% (n = 20) had BPH without prostatitis and 14% (n = 7) had a prostate carcinoma. While 34% (n = 17) of the 50 people had a positive bacterial culture [Table 2], a negative culture result was found in 66% of the samples (n =33). Of the patients who had a BPH with prostatitis (n =23), 10 Patients had a positive bacterial culture (43,5%). Of the patients who had a BPH without prostatitis (n =20), 4 Patients had a positive bacterial culture (20%). Of the patients diagnosed with prostate cancer (n =7), 3 Patients had a positive bacterial culture (42,9%). Hypothesis 1: positive bacterial cultures of the prostate gland are associated with a higher PSA and a larger prostate volume in patients, with or without histopathological signs of prostatitis, excluding patients with prostate cancer. A significant result was obtained for PSA values (r = 0,35, p = 0,02) as well as for the prostate volume (r = 0,43, p = 0,002). Persons with positive bacterial cultures have higher PSA values and have a greater prostate volume. This is significant in patients with and without histopathological signs of prostatitis. Hypothesis 1 can thus be confirmed [Figure 1, Figure 2, Table 1].
Hypothesis 2a: positive bacterial cultures of the prostate are associated with a higher PSA and a larger prostate volume in patients without histopathological signs of prostatitis. There were significant results for the PSA values (r = 0,70, p <0,001) as well as for the prostate volume (r = 0,49, p = 0,01). People with positive bacterial findings have higher PSA values and larger prostate volume in patients without histopathological signs of prostatitis. The results confirm the significant correlation, so hypothesis 2a can be accepted. 6
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Hypothesis 2b: positive bacterial cultures of the prostate are associated with higher PSA levels and larger prostate volumes in patients with histopathological signs of prostatitis. While the PSA values did not show a significant correlation with the prostate tissue culture (r = 0,15, p = 0,26) in patients with histopathological signs of prostatitis, a significant correlation was found with the prostate volume (r = 0,51, p = 0,01) in patients with histopathological signs of prostatitis. Correspondingly, hypothesis 2b can only be accepted with restrictions. Hypothesis 2c: bacterial cultures of the prostate are associated with higher PSA levels and a larger prostate volume in patients with prostate cancer. This did not produce any significant results for PSA levels (r = 0,4, p = 0,47), nor for prostate volume (r = 0,0, p = 0,50) in patients with prostate cancer. The results indicate that hypothesis 2c cannot be assumed to be valid. However, since in this case only few patient data were available, we realize that this assumption is statistically underpowered. It is quite possible that an effect exists, but the existing data are not sufficient to validate this. Hypothesis 3: positive bacterial cultures are associated with larger prostate volume in patients with or without histopathological signs of prostatitis, including patients with prostate cancer. There was a significant result for the prostate volume (r = 0,38, p = 0,004). People with positive bacterial findings have a higher prostate volume in comparison to the healthy male population. The correlation is significant, hypothesis 3 is thus to be confirmed.
Comment:
7
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First of all, a note on the ultrasound examination. We realize that some authors prefer transrectal ultrasound (TRUS) for prostate volume measurement. We opted for suprapubic, transabdominal ultrasound (TAUS) however for several reasons. Even though
transrectal
ultrasound
has
shown
to
be
superior
to
suprapubic
(transabdominal) volume measurement [16,17], TAUS is a widely accepted method of imaging the prostate worldwide. This is because it is more convenient for the patient and the urologist. TRUS on the other hand is associated with some level of resistance due to its invasiveness and associated discomfort [18]. Huang confirmed in 2004 that no statistically significant differences were found between the transabdominal ultrasound as compared to transrectal sonography [19].
The strength of our study are the easily reproducible results obtained by simple and safe examination methods. Our study shows that the bacterial colonization correlates strongly with a higher prostate gland volume in general. There is also a correlation concerning PSA levels, but in patients with histopathological signs of prostatitis without a positive tissue culture, PSA is elevated, too. As already mentioned in the introduction, it is well known the PSA correlates with prostate volume [7]. The correlation between prostate volume and positive tissue cultures has not been described in this form yet, but does not necessarily prove causation. Although this might be the case, the correlations noted in our study could also reflect a competing hypothesis that BPH growth leads to higher levels of PSA and that BPH growth makes the prostate a larger target for infection. At this stage, the uncertainty remains whether bacterial colonization is a consequence of enlargement, or whether it is the main cause. Further investigation into causation will require additional studies.
8
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Patients with chronic inflammation and benign prostatic hyperplasia (BPH) have been shown to have larger prostate volumes, with more severe lower urinary tract symptoms (LUTS) and a higher probability of acute urinary retention than their counterparts without inflammation [20]. Although the pathogenesis of BPH is not yet fully understood and several mechanisms seem to be involved in the development and progression, recent studies strongly suggest that BPH is an immune inflammatory disease. The T-cell activity and associated autoimmune reaction seem to induce epithelial and stromal cell proliferation [21]. In this context, the hypothesis published by Roper about the development of BPH opens an interesting field for discussion: the enzyme Phospholipase D (PLD) which is attached to the outer membrane of Escherichia coli (E. coli) is the basic cause of BPH. It has been asserted that repeated colonization by E. coli of the transitional zone of the prostate gland and the release of PLD following repeated destruction of these colonized bacteria leads to prostate enlargement. Also involved are a variety of interleukins and other inflammatory cell cytokines, secreted by the stroma, which may further promote autocrine/paracrine proliferation of BPH cells. [22]. Several different stimuli may induce chronic prostatic inflammation, which in turn could lead to tissue damage and continuous wound healing, thus contributing to prostatic enlargement. In our study cohort, lack of proof of bacterial colonization does not necessarily prove the absence of bacterial colonization as such. Some bacteria probably can´t be detected conventionally. Interestingly, in 2012, a study group reported that they had detected bacteria in women's urine, which remained unobserved in standard tests. The standard urine culture does not detect slow-growing bacteria that will not survive in the presence of oxygen. In contrast to previously known germs, which are routinely found in urine samples, for example, in the case of urinary bladder infections, these 9
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bacteria would not grow in the laboratory under normal conditions, which explains why they have been overlooked in urine analyzes. They detected the microorganisms by genetic analysis [23]. In addition to our findings, an enlarged prostate can also be seen in patients with positive prostate tissue culture, even in the absence of inflammatory signs of the tissue. Thus, the inflammatory stimulus predescribed in the cited studies above cannot be exclusively responsible for an enlargement of the prostate gland. Other pathways of liberation of growth factors due to bacterial colonization might also be responsible. So far, no study data exists whether certain types of bacteria might have a positive, negative or no effect on the prostate gland and the direct connection between bacterial colonization and clinical symptoms is yet to be made. Recently, a study group from Milano, Italy, states the importance of the prostatic microbiome of the prostate tumor microenvironment, which contains a plethora of bacteria. The bacteria set themselves within the gland with a distribution dependent on the nature of the tissue, thus suggesting a possible pathophysiological correlation between the composition of the local microbial niche and the presence of the tumor itself. The role of specific bacteria and their potential to be exploited as new biomarkers remains to be investigated further [24] and could be a predictor of response to treatment as well. Thus, the ability to identify patients with a high risk for BPH progression and cancer genesis may be crucial to prevent aggravation of symptoms, genesis of prostate cancer and eventually develop target therapies. The ability of the immune system to fight diseases could depend on the urogenital microbiome. Although research on the urogenital tract are still scarce, initial studies have shown that the microbial populations in urine, seminal fluid, and prostatic fluid are significantly different in men with BPH from men with prostate cancer. An important goal for the future is to learn more about its role in disease development by 10
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researching the microbiome of the genitourinary tract. Some particular colonization types might even offer protection against recurrent infections, prostate enlargement, LUTS or even cancer. Keeping in mind the rapidly growing antibiotic resistance worldwide, it is important to identify true uropathogens to be able to optimize antibacterial therapy.
11
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Conclusions: People with positive bacterial prostatic tissue culture have a higher prostate volume in comparison to patients with negative culture findings. Persons with positive bacterial prostate tissue cultures show a tendency towards higher PSA levels.
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References: [1] Berry SJ, Coffey DS, Walsh PC, et al. The development of human benign prostatic hyperplasia with age. J Urol 1984;132:474–9. [2] Xia SJ, Xu XX, Teng JB, et al. Characteristic pattern of human prostatic growth with age. Asian J Androl 2002;4:269–71. [3] Emberton M, Andriole GL, de la Rosette J, et al. Benign prostatic hyperplasia: a progressive disease of aging men. Urology 2003;61:267–73. [4] Bosch JL, Tilling K, Bohnen AM, et al. Establishing normal reference ranges for prostate volume change with age in the population-based Krimpen-study: prediction of future prostate volume in individual men. Prostate 2007;67:1816–24. [5] Rhodes T, Girman CJ, Jacobsen SJ, et al. Longitudinal prostate growth rates during 5 years in randomly selected community men 40 to 79 years old. J Urol 1999;161:1174–9. [6] Loeb S, Kettermann A, Carter HB, et al. Prostate volume changes over time: results from the Baltimore Longitudinal Study of Aging. J Urol 2009;182:1458–62. [7] Roehrborn CG, Boyle P, Gould AL, et al. Serum prostate-specific antigen as a predictor of prostate volume in men with benign prostatic hyperplasia. Urology 1999;53:581-9. [8] Fibbi B, Penna G, Morelli A, et al. Chronic inflammation in the pathogenesis of benign prostatic hyperplasia. Int J Androl 2010;33:475-88.
13
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[9] De Nunzio C, Kramer G, Marberger M, et al. The controversial relationship between benign prostatic hyperplasia and prostate cancer: the role of inflammation. Eur Urol 2011;60:106-17. [10] De Marzo AM, Platz EA, Sutcliffe S, et al. Inflammation in prostate carcinogenesis. Nat Rev Cancer 2007;7:256–269. [11] Vasto S, Carruba G, Candore G, et al. Inflammation and prostate cancer. Future Oncol 2008;4:637–645. [12] Cohen RJ, Shannon BA, McNeal JE, et al. Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: a possible link to cancer evolution? J Urol 2005;173:1969–1974. [13] Alexeyev OA, Marklund I, Shannon B, et al. Direct visualization of Propionibacterium acnes in prostate tissue by multicolor fluorescent in situ hybridization assay. J Clin Microbiol 2007;45:3721–3728. [14] Heidler S, Bretterbauer K, Schwarz S, et al. Diversity of Bacterial Urine and Prostate Gland Tissue Cultures in Patients Undergoing Transurethral Prostate Gland Resection. Urol Int 2016;97:336-339. [15] Aarnink RG, de la Rosette JJ, Debruyne FM, et al. Formula-derived prostate volume determination. Eur Urol 1996;29:399–402. [16] Loch AC, Bannowsky A, Baeurle L, et al. Technical and anatomical essentials for transrectal ultrasound of the prostate. World J Urol 2007;25: 361.
14
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[17] Stravodimos KG, Petrolekas A, Kapetanakis T, et al. TRUS versus transabdominal ultrasound as a predictor of enucleated adenoma weight in patients with BPH: a tool for standard preoperative work-up? Int Urol Nephrol 2009;41:767. [18] Park SB, Kim JK, Choi SH, et al. Prostate volume measurement by TRUS using heights obtained by transaxial and midsagittal scanning: comparison with specimen volume following radical prostatectomy. Korean J Radiol 2000;1:110-3. [19] Huang Foen Chung JW, de Vries SH, Raaijmakers R, Postma R et al. Prostate volume ultrasonography: the influence of transabdominal versus transrectal approach, device type and operator. Eur Urol 2004;46:352-6. [20] Gandaglia G, Briganti A, Gontero P, et al. The role of chronic prostatic inflammation in the pathogenesis and progression of benign prostatic hyperplasia (BPH). BJU Int 2013;112:432-41. [21] Bostanci Y, Kazzazi A, Momtahen S, Laze J, et al. Correlation between benign prostatic hyperplasia and inflammation.Curr Opin Urol 2013;23:5-10. [22] Roper WG. The prevention of benign prostatic hyperplasia (bph).Med Hypotheses 2017 Mar;100:4-9. [23] Wolfe AJ, Toh E, Shibata N, et al. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol 2012;50:1376-83. [24] Cavarretta I, Ferrarese R, Cazzaniga W, et al. The Microbiome of the Prostate Tumor Microenvironment. Eur Urol 2017;72:625-631.
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Figure 1: PSA and positive prostate gland tissue culture Figure 2: prostate volume and positive prostate gland tissue culture
16
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Table 1: study group data All Patients (n=50)
Prostatitis (n=30)
No prostatitis (n=20)
Age
Vol.
PSA
Age
Vol.
PSA
Age
Vol.
PSA
Positive tissue culture (n=17) Age Vol. PSA
Negative tissue culture (n=33) Age Vol. PSA
Mean (M)
70,9
49,9
4,2
72.2
43.4*
3.9
69.3
55.6*
2.88*
72.7
62.3*
4.9
70.2
42.9
2.9
Median (Mdn) Stan-darddevi-ation (SD) Mini-mum (Min) Maxi-mum (Max) Vol.: *p<.05:
71,5
50
3,2
73.0
40.0
3.4
68.0
55.0
2.15
74
50
4.3
70
40
2.2
9,1
22,6
3,9
8.6
22.5
3.2
8.2
23.7
2.22
8.8
28.4
3.1
8.4
19.1
2.6
48
15
0,2
59.0
15.0
0.4
60.0
20.0
0.50
59
15
0.6
59
20
0.4
88
100
17,7
88.0
100
12
86
100
9.30
87
100
9.9
88
80
12
prostate volume statistical significance
17
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Table 2: Types of bacteria in prostate gland tissue Patients Bacteria 1
Staphylococcus saprophyticus
2
Escherichia coli
3
Staphylococcus aureus
4
Pseudomonas aeruginosa
5
Enterococcus faecalis
6
Staphylococcus epidermidis
7
Corynebakterium Glucuronolyticum
8
Escherichia coli (AmpC) - MRE
9
Staphylococcus aureus
10
Enterobacter cloace
11
Staphylococcus aureus
12
Corynebacterium glucuronolyticum
13
Enterococcus faecalis
14
Staphylococcus epidermidis and Koagulase negativ
15
Enterococcus faecalis
16
Staphylococcus epidermidis
17
Escherichia coli
18
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Figure 1
20
15
PSA
10
5
0 positiv e
negativ prostatic tissue culture
19
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Figure 2
100
80
60 Prostate volume 40
20
0 positiv e
negativ prostatic tissue culture
20
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